Project Summary (Rajan Project) CRISPR-Cas systems are comprised of RNA-protein complexes that inactivate foreign DNA or RNA entering a bacterial or archaeal cell by producing sequence specific cleavage in the intruding DNA or RNA. CRISPR was given the status of a ?bacterial/archaeal immune system? because of its ability to protect against recurring phage infections, based on genetic memory retained from the past infections. The RNA-based DNA targeting function of CRISPR-Cas systems is widely used for gene editing and has promise for the treatment of inherited and infectious diseases. One of the less characterized aspects of CRISPR-Cas systems is their role in alternate functions in bacteria other than phage predation. For example, CRISPR-Cas systems have been shown to enhance bacterial pathogenicity in Francisella tularensis (Ft) novicida, Neisseria meningitidis, and Campylobacter jejuni. Paradoxically, in highly drug resistant bacteria such as Staphylococcus aureus and Francisella tularensis, components of the CRISPR-Cas systems are inactivated possibly to capitalize on the benefits of horizontal gene transfer. It is critical that we understand how bacteria turn on and off the benefits of CRISPR-Cas systems. The proposed work aims to characterize the biochemical, structural, and functional mechanisms by which the CRISPR-Cas complexes of Ft. novicida enhance bacterial pathogenicity by down- regulating a bacterial lipoprotein (BLP) essential for host recognition and immune response. A non-canonical, RNA-independent, Mn2+-specific DNA cleavage activity for Cas9 and Cpf1 (two Ft. novicida Cas proteins) was observed recently in the PL's lab. The structural and conformational basis by which Cas9 and Cpf1 differentiate RNA-DNA, RNA-RNA, and DNA-Mn2+ as their substrates will be further characterized using a combination of in vitro and in vivo assays, X-ray crystallography, and Small Angle X-ray Scattering. The results from the proposed studies will identify the nuclease involved in blp mRNA degradation and the mechanistic aspects of how Cas9 associates with other cellular components for regulating bacterial pathogenicity. The basis of substrate recognition by Cas9 and Cpf1 will provide mechanistic details of a previously unknown activity of these proteins and how these enzymes perform RNA-independent DNA cleavage. The results will provide the basis of future studies on characterizing CRISPR-Cas mediated pathogenicity in other bacteria and the physiological relevance of RNA-independent, Mn2+-dependent DNA cleavage in bacterial physiology. This information can be used to develop innovative strategies for combating microbial antibiotic resistance and development of anti-microbials.